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BACKGROUND: Asialoglycoprotein receptor 1 (ASGR1), primarily expressed on hepatocytes, promotes the clearance and the degradation of glycoproteins, including lipoproteins, from the circulation. In humans, loss-of-function variants of ASGR1 are associated with a favorable metabolic profile and reduced incidence of cardiovascular diseases. The molecular mechanisms by which ASGR1 could affect the onset of metabolic syndrome and obesity are unclear. Therefore, here we investigated the contribution of ASGR1 in the development of metabolic syndrome and obesity. METHODS: ASGR1 deficient mice (ASGR1-/-) were subjected to a high-fat diet (45% Kcal from fat) for 20 weeks. The systemic metabolic profile, hepatic and visceral adipose tissue were characterized for metabolic and structural alterations, as well as for immune cells infiltration. RESULTS: ASGR1-/- mice present a hypertrophic adipose tissue with 41% increase in fat accumulation in visceral adipose tissue (VAT), alongside with alteration in lipid metabolic pathways. Intriguingly, ASGR1-/- mice exhibit a comparable response to an acute glucose and insulin challenge in circulation, coupled with notably decreased in circulating cholesterol levels. Although the liver of ASGR1-/- have similar lipid accumulation to the WT mice, they present elevated levels of liver inflammation and a decrease in mitochondrial function. CONCLUSION: ASGR1 deficiency impacts energetic homeostasis during obesity leading to improved plasma lipid levels but increased VAT lipid accumulation and liver damage.
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Receptor de Asialoglicoproteína , Síndrome Metabólica , Animais , Humanos , Camundongos , Tecido Adiposo/metabolismo , Receptor de Asialoglicoproteína/genética , Dieta Hiperlipídica , Inflamação/metabolismo , Lipídeos , Fígado/metabolismo , Síndrome Metabólica/complicações , Camundongos Endogâmicos C57BL , Obesidade/complicaçõesRESUMO
AIMS: Mitochondria are plastic organelles that continuously undergo biogenesis, fusion, fission, and mitophagy to control cellular energy metabolism, calcium homeostasis, hormones, sterols, and bile acids (BAs) synthesis. Here, we evaluated how the impairment of mitochondrial fusion in hepatocytes affects diet-induced liver steatosis and obesity. METHODS AND RESULTS: Male mice selectively lacking the key protein involved in inner mitochondrial fusion, optic atrophy 1 (OPA1) (OPA1ΔHep) were fed a high fat diet (HFD) for 20 weeks. OPA1ΔHep mice were protected from the development of hepatic steatosis and obesity because of reduced lipid absorption; a profile which was accompanied by increased respiratory exchange ratio in vivo, suggesting a preference for carbohydrates in OPA1ΔHep compared to controls. At the molecular level, this phenotype emerged as a consequence of poor mitochondria-peroxisome- endoplasmic reticulum (ER) tethering in OPA1 deficient hepatocytes, which impaired BAs conjugation and release in the bile, thus impacting lipid absorption from the diet. Concordantly, the liver of subjects with non-alcoholic fatty liver disease (NAFLD) presented an increased expression of OPA1 and of the network of proteins involved in mitochondrial function when compared with controls. CONCLUSION: Patients with NAFLD present increased expression of proteins involved in mitochondrial fusion in the liver. The selective deficency of OPA1 in hepatocytes protects mice from HFD-induced metabolic dysfunction by reducing BAs secretion and dietary lipids absorption as a consequence of reduced liver mitochondria-peroxisome-ER tethering.
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Hepatopatia Gordurosa não Alcoólica , Humanos , Masculino , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/complicações , Dinâmica Mitocondrial , Fígado/metabolismo , Hepatócitos/metabolismo , Obesidade/metabolismo , Dieta Hiperlipídica , Lipídeos , Metaboloma , Ácidos e Sais Biliares/metabolismo , Camundongos Endogâmicos C57BLRESUMO
AIMS: Inflammatory pathways and immune system dysregulation participate in the onset and progression of cardiometabolic diseases. The dendritic cell immunoreceptor 2 (DCIR2) is a C-type lectin receptor mainly expressed by conventional type 2 dendritic cells, involved in antigen recognition and in the modulation of T cell response. Here, we investigated the effect of DCIR2 deficiency during the development of obesity. METHODS: DCIR2 KO mice and the WT counterpart were fed with high-fat diet (HFD) for 20 weeks. Weight gain, glucose and insulin tolerance were assessed, parallel to immune cell subset profiling and histological analysis. RESULTS: After HFD feeding, DCIR2 KO mice presented altered conventional dendritic cell distribution within the liver without affecting markers of hepatic inflammation. These observations were liver restricted, since immune profile of metabolic and lymphoid organs-namely adipose tissue, spleen and mesenteric lymph nodes-did not show differences between the two groups. This reflected in a similar metabolic profile of DCIR2 KO compared to WT mice, characterized by comparable body weight gain as well as adipose tissues, spleen, Peyer's patches and mesenteric lymph nodes weight at sacrifice. Also, insulin response was similar in both groups. CONCLUSION: Our data show that DCIR2 has a redundant role in the progression of diet-induced obesity and inflammation.
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Insulinas , Obesidade , Animais , Camundongos , Células Dendríticas , Dieta Hiperlipídica/efeitos adversos , Inflamação/metabolismo , Insulinas/metabolismo , Fígado/patologia , Obesidade/etiologia , Obesidade/patologiaRESUMO
Background and aim: The primary transcript of fibronectin (FN) undergoes alternative splicing to generate different isoforms, including FN containing the Extra Domain A (FN_EDA+), whose expression is regulated spatially and temporarily during developmental and disease conditions including acute inflammation. The role of FN_EDA+ during sepsis, however, remains elusive. Methods: Mice constitutively express the EDA domain of fibronectin (EDA+/+); lacking the FN EDA domain (EDA-/-) or with a conditional ablation of EDA + inclusion only in liver produced FN (alb-CRE+EDA floxed mice) thus expressing normal plasma FN were used. Systemic inflammation and sepsis were induced by either LPS injection (70 mg/kg) or by cecal ligation and puncture (CLP) Neutrophils isolated from septic patients were tested for neutrophil binding ability. Results: We observed that EDA+/+ were protected toward sepsis as compared to EDA-/- mice. Also alb-CRE+EDA floxed mice presented reduced survival, thus indicating a key role for EDA in protecting toward sepsis. This phenotype was associated with improved liver and spleen inflammatory profile. Ex vivo experiments showed that neutrophils bind to a larger extent to an FN_EDA + coated surface as compared to FN, thus potentially limiting their over-reactivity. Conclusions: Our study demonstrates that the inclusion of the EDA domain in fibronectin dampens the nflammatoryi consequences of sepsis.
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BACKGROUND: High fat diet (HFD) chronically hyper-activates the myeloid cell precursors, but whether it affects the neutrophil aging is unknown. PURPOSE: We characterized how HFD impacts neutrophil aging, infiltration in metabolic tissues and if this aging, in turn, modulates the development of metabolic alterations. We immunophenotyped neutrophils and characterized the metabolic responses in physiology (wild-type mice, WT) and in mice with constitutively aged neutrophils (MRP8 driven conditional deletion of CXCR4; herein CXCR4fl/flCre+) or with constitutively fresh neutrophils (MRP8 driven conditional deletion of CXCR2; CXCR2fl/flCre+), following 20 weeks of HFD feeding (45 % kcal from fat). FINDINGS: After 20 weeks HFD, the gluco-metabolic profile of CXCR4fl/flCre+ mice was comparable to that of WT mice, while CXCR2fl/flCre+ mice were protected from metabolic alterations. CXCR4fl/flCre+ infiltrated more, but CXCR2fl/flCre+ neutrophils infiltrated less, in liver and visceral adipose tissue (VAT). As consequence, while CXCR4fl/flCre+ resulted into hepatic "suicidal" neutrophils extracellular traps (NETs) and altered immune cell architecture in VAT, CXCR2fl/flCre+ promoted proresolutive hepatic NETs and reduced accumulation of pro-inflammatory macrophages in VAT. In humans, higher plasma levels of Cxcl12 (CXCR4 ligand) correlated with visceral adiposity while higher levels of Cxcl1 (the ligand of CXCR2) correlated with indexes of hepatic steatosis, adiposity and metabolic syndrome. CONCLUSIONS: Neutrophil aging might contribute to the development of HFD induced metabolic disorders.
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Dieta Hiperlipídica , Neutrófilos , Humanos , Camundongos , Animais , Idoso , Neutrófilos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ligantes , Modelos Animais de Doenças , Envelhecimento , Camundongos Endogâmicos C57BLRESUMO
Background and aims: Atherogenesis results from altered lipid metabolism and impaired immune response. Emerging evidence has suggested that dendritic cells (DCs) participate to atherosclerosis-related immune response, but their impact is scarcely characterized. Clec4a4 or DCIR2 (Dendritic cell immunoreceptor 2) is a C-type lectin receptor, mainly expressed by CD8α- DCs, able to modulate T cell immunity. However, whether this DC subset could play a role in the atherogenesis is still poorly understood. Thus, the aim of this study is to investigate whether the absence of Clec4a4 could affect atherosclerosis-related immune response and atherosclerosis itself. Methods: Dcir2 -/- Ldlr -/- and Ldlr -/- mice were fed a standard diet or cholesterol-enriched diet for 12 weeks. Subsequently, the profile of circulating and lymph nodes-resident immune cells was investigated together with the analysis of plasma lipid levels and atherosclerotic plaque extension in the aorta. Results: Here, we show that Clec4a4 expression is downregulated under hypercholesterolemia and its deficiency in Ldlr -/- mice results in the reduction of atherosclerotic plaque formation, together with altered lipid metabolism and impaired myeloid immune cell distribution. Conclusions: Our findings suggest a pro-atherosclerotic role of Clec4a4 in experimental atherosclerosis.
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Activation of T cells relies on the availability of intracellular cholesterol for an effective response after stimulation. We investigated the contribution of cholesterol derived from extracellular uptake by the low-density lipoprotein (LDL) receptor in the immunometabolic response of T cells. By combining proteomics, gene expression profiling, and immunophenotyping, we described a unique role for cholesterol provided by the LDLR pathway in CD8+ T cell activation. mRNA and protein expression of LDLR was significantly increased in activated CD8+ compared to CD4+ WT T cells, and this resulted in a significant reduction of proliferation and cytokine production (IFNγ, Granzyme B, and Perforin) of CD8+ but not CD4+ T cells from Ldlr -/- mice after in vitro and in vivo stimulation. This effect was the consequence of altered cholesterol routing to the lysosome resulting in a lower mTORC1 activation. Similarly, CD8+ T cells from humans affected by familial hypercholesterolemia (FH) carrying a mutation on the LDLR gene showed reduced activation after an immune challenge.
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Linfócitos T CD8-Positivos , Colesterol , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Receptores de LDL , Animais , Linfócitos T CD8-Positivos/metabolismo , Colesterol/metabolismo , Citocinas/metabolismo , Granzimas/metabolismo , Humanos , Hiperlipoproteinemia Tipo II , Interferon gama/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Perforina , RNA Mensageiro/genética , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
BACKGROUND: Cholesterol is central to pancreatic ß-cell physiology and alterations of its homeostasis contribute to ß-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane. PCSK9 is also expressed in the pancreas and loss of function mutations of PCSK9 result in higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Aim of this study was to investigate whether PCSK9 also impacts ß-cells function. METHODS: Pancreas-specific Pcsk9 null mice (Pdx1Cre/Pcsk9 fl/fl) were generated and characterized for glucose tolerance, insulin release and islet morphology. Isolated Pcsk9-deficient islets and clonal ß-cells (INS1E) were employed to characterize the molecular mechanisms of PCSK9 action. RESULTS: Pdx1Cre/Pcsk9 fl/fl mice exhibited normal blood PCSK9 and cholesterol levels but were glucose intolerant and had defective insulin secretion in vivo. Analysis of PCSK9-deficient islets revealed comparable ß-cell mass and insulin content but impaired stimulated secretion. Increased proinsulin/insulin ratio, modifications of SNARE proteins expression and decreased stimulatedcalcium dynamics were detected in PCSK9-deficient ß-cells. Mechanistically, pancreatic PCSK9 silencing impacts ß-cell LDLR expression and cholesterol content, both in vivo and in vitro. The key role of LDLR is confirmed by the demonstration that LDLR downregulation rescued the phenotype. CONCLUSIONS: These findings establish pancreatic PCSK9 as a novel critical regulator of the functional maturation of the ß-cell secretory pathway, via modulation of cholesterol homeostasis.
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Diabetes Mellitus Tipo 2 , Pró-Proteína Convertase 9 , Animais , Glicemia/metabolismo , Cálcio/metabolismo , Colesterol , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Proinsulina/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas SNARE/metabolismo , Via Secretória , Serina Endopeptidases/genética , Subtilisinas/metabolismoRESUMO
BACKGROUND: HDL is endowed with several metabolic, vascular, and immunoinflammatory protective functions. Among them, a key property is to promote reverse cholesterol transport from cells back to the liver. The aim of this study was to estimate the association of scavenger receptor class B type I (SR-BI)- and ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux (the two major routes for cholesterol efflux to HDL) with the presence, extent, and severity of coronary artery disease (CAD), vascular wall remodelling processes, coronary plaque characteristics, and the incidence of myocardial infarction in the different subgroups of patients from the CAPIRE study. METHODS: Patients (n = 525) from the CAPIRE study were divided into two groups: low-risk factors (RF), with 0-1 RF (n = 263), and multiple-RF, with ≥2 RFs; within each group, subjects were classified as no-CAD or CAD based on the segment involvement score (SIS) evaluated by coronary computed tomography angiography (SIS = 0 and SIS > 5, respectively). SR-BI- and ABCA1-mediated cholesterol efflux were measured using the plasma of all patients. RESULTS: SR-BI-mediated cholesterol efflux was significantly reduced in patients with CAD in both the low-RF and multiple-RF groups, whereas ABCA1-mediated cholesterol efflux was similar among all groups. In CAD patients, multivariable analysis showed that SR-BI-mediated cholesterol efflux <25th percentile predicted cardiovascular outcome (odds ratio 4.1; 95% CI: 1.3-13.7; p = .019), whereas ABCA-1-mediated cholesterol efflux and HDL-C levels significantly did not. Despite this finding, reduced SR-BI-mediated cholesterol efflux was not associated with changes in high-risk plaque features or changes in the prevalence of elevated total, non-calcified, and low-attenuation plaque volume. CONCLUSION: SR-BI-mediated cholesterol efflux capacity is lower in patients with diffuse coronary atherosclerosis. In addition, a lower SR-BI-mediated cholesterol efflux capacity is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features. Key MessagesIncreased cholesterol efflux capacity, an estimate of HDL function, is associated with a reduced CVD risk, regardless of HDL-C levels.HDL-C levels are significantly lower in patients with CAD.Lower SR-BI-mediated cholesterol efflux capacity is observed in patients with diffuse coronary atherosclerosis and is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features.
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Doença da Artéria Coronariana , Placa Aterosclerótica , Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol , HDL-Colesterol , Humanos , Lipoproteínas HDL/metabolismo , Placa Aterosclerótica/diagnóstico por imagem , Receptores Depuradores Classe B/metabolismoRESUMO
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association.
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AIMS: PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. METHODS AND RESULTS: Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. CONCLUSION: PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.
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Insuficiência Cardíaca , Pró-Proteína Convertase 9 , Animais , Insuficiência Cardíaca/genética , Masculino , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/genética , Receptores de LDL/genética , Volume SistólicoRESUMO
Aims: PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results: Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion: PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/genética , Intolerância à Glucose/metabolismo , Secreção de Insulina/fisiologia , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Animais , Apoptose , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
Cholesterol homeostasis has a pivotal function in regulating immune cells. Here we show that apolipoprotein E (apoE) deficiency leads to the accumulation of cholesterol in the cell membrane of dendritic cells (DC), resulting in enhanced MHC-II-dependent antigen presentation and CD4+ T-cell activation. Results from WT and apoE KO bone marrow chimera suggest that apoE from cells of hematopoietic origin has immunomodulatory functions, regardless of the onset of hypercholesterolemia. Humans expressing apoE4 isoform (ε4/3-ε4/4) have increased circulating levels of activated T cells compared to those expressing WT apoE3 (ε3/3) or apoE2 isoform (ε2/3-ε2/2). This increase is caused by enhanced antigen-presentation by apoE4-expressing DCs, and is reversed when these DCs are incubated with serum containing WT apoE3. In summary, our study identifies myeloid-produced apoE as a key physiological modulator of DC antigen presentation function, paving the way for further explorations of apoE as a tool to improve the management of immune diseases.
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Apresentação de Antígeno , Apolipoproteínas E/genética , Células Dendríticas/metabolismo , Ativação Linfocitária , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Animais , Apolipoproteína E4/genética , Células da Medula Óssea/citologia , Diferenciação Celular , Movimento Celular , Colesterol/metabolismo , Células Dendríticas/citologia , Ácidos Graxos/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Antígenos de Histocompatibilidade Classe II , Humanos , Hipercolesterolemia/metabolismo , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxisteróis/química , Oxisteróis/metabolismo , Fosfolipídeos/químicaRESUMO
BACKGROUND AND AIMS: Proprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury. METHODS: Carotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knock-out (PCSK9-/-) and wild type littermate (PCSK9+/+) mice. RESULTS: In PCSK9-/- mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9+/+ mice. When compared with PCSK9-/-, lesions of PCSK9+/+ mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9-/-, when compared to PCSK9+/+ cells, showed higher levels of α-smooth muscle actin (α-SMA; 2.24 ± 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 ± 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA(-54 ± 14%; p < 0.01). PCSK9-/- cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9-/- SMCs, led to a downregulation of α-SMA (-56 ± 2%; p < 0.01), MHC-II (-45% ± 25.5 fold: p = 0.06) and calponin (-25% ± 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 ± 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9-/- was significantly lower compared to PCSK9 reconstituted cells. CONCLUSIONS: Taken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall.
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Artérias Carótidas/patologia , Lesões das Artérias Carótidas/patologia , Neointima/patologia , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/fisiologia , Animais , Aorta/metabolismo , Becaplermina , Diferenciação Celular , Movimento Celular , Proliferação de Células , Quimiotaxia , LDL-Colesterol/metabolismo , Citoesqueleto/metabolismo , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Fenótipo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Túnica Íntima/patologiaRESUMO
The primary transcript of fibronectin undergoes alternative splicing in the cassette-type EDA and EDB exons and in the IIICs segment to generate different protein isoforms. Human carotid atherosclerotic plaques with a more stable phenotype are enriched with EDA containing fibronectin (FN-EDA). The aim of this study was to investigate the role of EDA containing fibronectin during atherogenesis. Mice constitutively expressing or lacking the EDA domain of fibronectin (EDA+/+ or EDA-/-)were crossed with ApoE-/- or LDL-R-/- mice and fed with a western type diet for 12 weeks. Lack of FN-EDA resulted in reduced atherosclerosis and in a plaque phenotype characterised by decreased calponin positive VSMC's (-15 %) and increased macrophages (+20 %). This was paralleled by increased MMP2, MMP9, and reduced TIMP2, collagen 1A1, 1A2 and 3A1 gene expression compared to that of wild-type and EDA+/+ mice. In vitro, VSMCs and macrophages isolated from EDA-/- miceshowed increased MMPs expression and activity compared to wild-type or EDA+/+ mice. Albumin-Cre recombinase/EDA+/+/ApoE-/- mice, which produceEDA containing FN only in peripheral tissues, presented an extension, a composition and a gene expression pattern in the atherosclerotic lesions similar to that of controls. The inclusion of EDA in FN results in larger atherosclerotic plaques compared to mice lacking EDA but with a more favourable phenotype in two animals models of atherosclerosis. This effect depends on the EDA-containing fibronectin produced by cells in the vasculature but not in the liver. These observations set the stage for investigating the properties of circulating EDA containing FN in improving plaque stability.
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Doenças da Aorta/metabolismo , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Fibronectinas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Receptores de LDL/deficiência , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Colágeno/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibronectinas/deficiência , Fibronectinas/genética , Genótipo , Macrófagos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Receptores de LDL/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , CalponinasRESUMO
OBJECTIVE: Familial hypobetalipoproteinemia (FHBL) is autosomal codominant disorder of lipoprotein metabolism characterized by low plasma levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5(th) percentile of the distribution in the population. Patients with the clinical diagnosis of homozygous FHBL (Ho-FHBL) are extremely rare and few patients have been characterized at the molecular level. Here we report the medical history and the molecular characterization of one paediatric patient with clinical features of Ho-FHBL. METHODS: A one month old infant with failure to thrive, severe hypocholesterolemia and acanthocytosis was clinically and genetically characterized. Molecular characterization of the proband and her parents was performed by direct sequencing of the APOB gene and functional role of the identified mutations was assessed by the minigene methodology. RESULTS: The proband was found carrying two novel splicing mutations of the APOB gene (c.3696+1G > C and c.3697-1G > A). CHOK1H8 cells expressing minigenes harbouring the mutations showed that these two mutations were associated with the retention of intron 23 and skipping of exon 24, resulting in two truncated apoB fragments of approximate size of 26-28 % of ApoB-100 and the total absence of apoB. CONCLUSION: We describe the first case of Ho-FHBL due to two splicing mutations affecting both the donor and the acceptor splice sites of the same intron of the APOB gene occurring in the same patient. The clinical management of the proband is discussed and a review of the clinical and genetic features of the published Ho-FHBL cases is reported.
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Apolipoproteína B-100/genética , Homozigoto , Hipobetalipoproteinemias/diagnóstico , Hipobetalipoproteinemias/genética , Mutação , Abetalipoproteinemia/genética , Adulto , Processamento Alternativo , Colesterol/sangue , LDL-Colesterol/sangue , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Íntrons , MasculinoRESUMO
Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolomeasconserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.
Assuntos
Ácido Araquidônico/metabolismo , Colesterol/metabolismo , Metaboloma , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Araquidonato 5-Lipoxigenase/metabolismo , Aspirina/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Ácidos e Sais Biliares/metabolismo , Células Cultivadas , Colesterol/sangue , HDL-Colesterol/sangue , HDL-Colesterol/metabolismo , Humanos , Leucotrienos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Cavéolas/metabolismo , HDL-Colesterol/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Membrana Celular/metabolismo , HDL-Colesterol/química , Células Endoteliais/metabolismo , Ativação Enzimática/genética , Humanos , Óxido Nítrico/metabolismo , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais/genética , VasodilataçãoRESUMO
Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is the main endothelial receptor for oxidized low density lipoprotein (OxLDL). LOX-1 is highly expressed in endothelial cells of atherosclerotic lesions, but also in macrophages and smooth muscle cells. LOX-1 expression is upregulated by several inflammatory cytokines (such as TNF-α), by oxidative stress, and by pathological conditions, such as dyslipidemia, hypertension, and diabetes. High density lipoprotein (HDL) possess several atheroprotective properties; however under pathological conditions associated with inflammation and oxidative stress, HDL become dysfunctional and exhibit pro-inflammatory properties. In vitro, HDL can be modified by 15-lipoxygenase, an enzyme overexpressed in the atherosclerotic lesions. Here we report that, after modification with 15-lipoxygenase, HDL(3) lose their ability to inhibit TNFα-induced LOX-1 expression in endothelial cells; in addition, 15LO-modified HDL(3) induce LOX-1 mRNA and protein expression and bind to LOX-1 with increased affinity compared to native HDL(3). Altogether these findings confirm that 15LO-modified HDL(3) possess a pro-atherogenic role.